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Pickup, David James

Overview:

Viral inhibition of host immune defenses
Many viruses have evolved mechanisms to protect themselves from host immune defenses. Among this group are the orthopoxviruses, whose members include smallpox virus, one of the deadliest of human viruses, and cowpox virus, the virus that Edward Jenner used to begin the eradication of smallpox.

One of the especially interesting features of theses viruses is their ability to interfere with a wide range of innate and adaptive immune responses to infection. For example, we have found that cowpox virus inhibits inflammation by suppressing the actions of cytokines controlling inflammatory processes. Moreover, the virus does this in several ways: by preventing the synthesis of cytokines; by interfering with normal cytokine-receptor interactions; and by inhibiting cytokine-signaling pathways.

Our main research objectives are to identify mechanisms of virus-host interaction leading to the modification or alteration of host functions. Our working model is that such interactions are amongst the most important factors in viral pathogenesis. In addition, knowledge of these virus-host interactions should help in the development of new vaccines and therapies for a variety of conditions associated with infectious diseases, inflammatory diseases, autoimmune diseases, cancers, and organ transplantation.

Development of improved viral vaccines
Several excellent vaccine platforms exist, but among these vaccinia virus vaccines have unusual potential for targeting multiple different pathogens because of the extraordinary capacity of these vectors to encode multiple foreign proteins. Replication-defective vaccinia vectors are extremely safe. However, this safety comes at a cost. Because only a small amount of antigen can be produced during the single cycle of viral replication, vectors of this type typically require high doses and multiple boosts to induce protective immune responses.  We are interested in finding ways to enhance the immunogenicity of these replication-defective vaccine viruses without compromising on safety.


Positions:

Associate Professor of Virology in the Department of Molecular Genetics and Microbiology

Molecular Genetics and Microbiology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Member of the Duke Human Vaccine Institute

Duke Human Vaccine Institute
School of Medicine

Education:

Ph.D. 1979

Ph.D. — National Institute for Medical Research, London

Grants:

Mucosal vaccination to protect against HIV-1 infection at mucosal sites

Administered By
Pathology
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
July 01, 2012
End Date
June 30, 2017

Large Scale Antibody and TCell Epitope Discovery

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
September 30, 2004
End Date
September 29, 2011

Viral Inhibition of Host Defenses

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 1995
End Date
August 31, 2002

Viral Inhibition Of Host Defenses

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 1992
End Date
August 31, 1999

Cancer Center Core Support Grant

Administered By
Medicine, Medical Oncology
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
September 01, 1976
End Date
December 31, 1998

Comprehensive Cancer Center Core Support Grant

Administered By
Medicine, Medical Oncology
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
September 01, 1976
End Date
December 31, 1998

Comprehensive Cancer Center Core Support Grant

Administered By
Medicine, Medical Oncology
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
September 01, 1976
End Date
December 31, 1998

The Inhibition Of Inflammatory Processes

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 1993
End Date
August 31, 1995

The Inhibition Of Inflammatory Processes

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 1992
End Date
August 31, 1995

Structure And Function Of Poxvirus Genes

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 1993
End Date
March 31, 1995

The Structure And Function Of Poxvirus Genes

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 1986
End Date
August 01, 1989
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Publications:

Combined HIV-1 Envelope Systemic and Mucosal Immunization of Lactating Rhesus Monkeys Induces a Robust Immunoglobulin A Isotype B Cell Response in Breast Milk.

Maternal vaccination to induce anti-HIV immune factors in breast milk is a potential intervention to prevent postnatal HIV-1 mother-to-child transmission (MTCT). We previously demonstrated that immunization of lactating rhesus monkeys with a modified vaccinia Ankara (MVA) prime/intramuscular (i.m.) protein boost regimen induced functional IgG responses in milk, while MVA prime/intranasal (i.n.) boost induced robust milk Env-specific IgA responses. Yet, recent studies have suggested that prevention of postnatal MTCT may require both Env-specific IgA and functional IgG responses in milk. Thus, to investigate whether both responses could be elicited by a combined systemic/mucosal immunization strategy, animals previously immunized with the MVA prime/i.n. boost regimen received an i.n./i.m. combined C.1086 gp120 boost. Remarkably, high-magnitude Env-specific IgA responses were observed in milk, surpassing those in plasma. Furthermore, 29% of vaccine-elicited Env-specific B cells isolated from breast milk were IgA isotype, in stark contrast to the overwhelming predominance of IgG isotype Env-specific B cells in breast milk of chronically HIV-infected women. A clonal relationship was identified between Env-specific blood and breast milk B cells, suggesting trafficking of that cell population between the two compartments. Furthermore, IgA and IgG monoclonal antibodies isolated from Env-specific breast milk B cells demonstrated diverse Env epitope specificities and multiple effector functions, including tier 1 neutralization, antibody-dependent cellular cytotoxicity (ADCC), infected cell binding, and inhibition of viral attachment to epithelial cells. Thus, maternal i.n./i.m. combined immunization is a novel strategy to enhance protective Env-specific IgA in milk, which is subsequently transferred to the infant via breastfeeding.Efforts to increase the availability of antiretroviral therapy to pregnant and breastfeeding women in resource-limited areas have proven remarkably successful at reducing HIV vertical transmission rates. However, more than 200,000 children are infected annually due to failures in therapy implementation, monitoring, and adherence, nearly half by postnatal HIV exposure via maternal breast milk. Intriguingly, in the absence of antiretroviral therapy, only 10% of breastfed infants born to HIV-infected mothers acquire the virus, suggesting the existence of naturally protective immune factors in milk. Enhancement of these protective immune factors through maternal vaccination will be a critical strategy to reduce the global pediatric AIDS epidemic. We have previously demonstrated that a high magnitude of HIV Env-specific IgA in milk correlates with reduced risk of infant HIV acquisition. In this study, we describe a novel HIV vaccine regimen that induces potent IgA responses in milk and therefore could potentially protect against breast milk HIV MTCT.

Authors
Nelson, CS; Pollara, J; Kunz, EL; Jeffries, TL; Duffy, R; Beck, C; Stamper, L; Wang, M; Shen, X; Pickup, DJ; Staats, HF; Hudgens, MG; Kepler, TB; Montefiori, DC; Moody, MA; Tomaras, GD; Liao, H-X; Haynes, BF; Ferrari, G; Fouda, GGA; Permar, SR
MLA Citation
Nelson, CS, Pollara, J, Kunz, EL, Jeffries, TL, Duffy, R, Beck, C, Stamper, L, Wang, M, Shen, X, Pickup, DJ, Staats, HF, Hudgens, MG, Kepler, TB, Montefiori, DC, Moody, MA, Tomaras, GD, Liao, H-X, Haynes, BF, Ferrari, G, Fouda, GGA, and Permar, SR. "Combined HIV-1 Envelope Systemic and Mucosal Immunization of Lactating Rhesus Monkeys Induces a Robust Immunoglobulin A Isotype B Cell Response in Breast Milk." Journal of virology 90.10 (May 2016): 4951-4965.
PMID
26937027
Source
epmc
Published In
Journal of virology
Volume
90
Issue
10
Publish Date
2016
Start Page
4951
End Page
4965
DOI
10.1128/jvi.00335-16

Extracellular Virions: The Advance Guard of Poxvirus Infections.

Authors
Pickup, DJ
MLA Citation
Pickup, DJ. "Extracellular Virions: The Advance Guard of Poxvirus Infections." PLoS pathogens 11.7 (July 2, 2015): e1004904-. (Review)
PMID
26135739
Source
epmc
Published In
PLoS pathogens
Volume
11
Issue
7
Publish Date
2015
Start Page
e1004904
DOI
10.1371/journal.ppat.1004904

Poxviruses

Authors
Pickup, DJ
MLA Citation
Pickup, DJ. "Poxviruses." In: Encyclopedia of Life Sciences (eLS). (2015). (Addendum)
Source
manual
Published In
In: Encyclopedia of Life Sciences (eLS).
Publish Date
2015
DOI
10.1002/9780470015902.a0001083.pub3

T cell inactivation by poxviral B22 family proteins increases viral virulence.

Infections with monkeypox, cowpox and weaponized variola virus remain a threat to the increasingly unvaccinated human population, but little is known about their mechanisms of virulence and immune evasion. We now demonstrate that B22 proteins, encoded by the largest genes of these viruses, render human T cells unresponsive to stimulation of the T cell receptor by MHC-dependent antigen presentation or by MHC-independent stimulation. In contrast, stimuli that bypass TCR-signaling are not inhibited. In a non-human primate model of monkeypox, virus lacking the B22R homologue (MPXVΔ197) caused only mild disease with lower viremia and cutaneous pox lesions compared to wild type MPXV which caused high viremia, morbidity and mortality. Since MPXVΔ197-infected animals displayed accelerated T cell responses and less T cell dysregulation than MPXV US2003, we conclude that B22 family proteins cause viral virulence by suppressing T cell control of viral dissemination.

Authors
Alzhanova, D; Hammarlund, E; Reed, J; Meermeier, E; Rawlings, S; Ray, CA; Edwards, DM; Bimber, B; Legasse, A; Planer, S; Sprague, J; Axthelm, MK; Pickup, DJ; Lewinsohn, DM; Gold, MC; Wong, SW; Sacha, JB; Slifka, MK; Früh, K
MLA Citation
Alzhanova, D, Hammarlund, E, Reed, J, Meermeier, E, Rawlings, S, Ray, CA, Edwards, DM, Bimber, B, Legasse, A, Planer, S, Sprague, J, Axthelm, MK, Pickup, DJ, Lewinsohn, DM, Gold, MC, Wong, SW, Sacha, JB, Slifka, MK, and Früh, K. "T cell inactivation by poxviral B22 family proteins increases viral virulence." PLoS pathogens 10.5 (May 15, 2014): e1004123-.
PMID
24832205
Source
epmc
Published In
PLoS pathogens
Volume
10
Issue
5
Publish Date
2014
Start Page
e1004123
DOI
10.1371/journal.ppat.1004123

Mucosal immunization of lactating female rhesus monkeys with a transmitted/founder HIV-1 envelope induces strong Env-specific IgA antibody responses in breast milk.

We previously demonstrated that vaccination of lactating rhesus monkeys with a DNA prime/vector boost strategy induces strong T-cell responses but limited envelope (Env)-specific humoral responses in breast milk. To improve vaccine-elicited antibody responses in milk, hormone-induced lactating rhesus monkeys were vaccinated with a transmitted/founder (T/F) HIV Env immunogen in a prime-boost strategy modeled after the moderately protective RV144 HIV vaccine. Lactating rhesus monkeys were intramuscularly primed with either recombinant DNA (n = 4) or modified vaccinia virus Ankara (MVA) poxvirus vector (n = 4) expressing the T/F HIV Env C.1086 and then boosted twice intramuscularly with C.1086 gp120 and the adjuvant MF59. The vaccines induced Env-binding IgG and IgA as well as neutralizing and antibody-dependent cellular cytotoxicity (ADCC) responses in plasma and milk of most vaccinated animals. Importantly, plasma neutralization titers against clade C HIV variants MW965 (P = 0.03) and CAP45 (P = 0.04) were significantly higher in MVA-primed than in DNA-primed animals. The superior systemic prime-boost regimen was then compared to a mucosal-boost regimen, in which animals were boosted twice intranasally with C.1086 gp120 and the TLR 7/8 agonist R848 following the same systemic prime. While the systemic and mucosal vaccine regimens elicited comparable levels of Env-binding IgG antibodies, mucosal immunization induced significantly stronger Env-binding IgA responses in milk (P = 0.03). However, the mucosal regimen was not as potent at inducing functional IgG responses. This study shows that systemic MVA prime followed by either intranasal or systemic protein boosts can elicit strong humoral responses in breast milk and may be a useful strategy to interrupt postnatal HIV-1 transmission.

Authors
Fouda, GGA; Amos, JD; Wilks, AB; Pollara, J; Ray, CA; Chand, A; Kunz, EL; Liebl, BE; Whitaker, K; Carville, A; Smith, S; Colvin, L; Pickup, DJ; Staats, HF; Overman, G; Eutsey-Lloyd, K; Parks, R; Chen, H; Labranche, C; Barnett, S; Tomaras, GD; Ferrari, G; Montefiori, DC; Liao, H-X; Letvin, NL; Haynes, BF; Permar, SR
MLA Citation
Fouda, GGA, Amos, JD, Wilks, AB, Pollara, J, Ray, CA, Chand, A, Kunz, EL, Liebl, BE, Whitaker, K, Carville, A, Smith, S, Colvin, L, Pickup, DJ, Staats, HF, Overman, G, Eutsey-Lloyd, K, Parks, R, Chen, H, Labranche, C, Barnett, S, Tomaras, GD, Ferrari, G, Montefiori, DC, Liao, H-X, Letvin, NL, Haynes, BF, and Permar, SR. "Mucosal immunization of lactating female rhesus monkeys with a transmitted/founder HIV-1 envelope induces strong Env-specific IgA antibody responses in breast milk." J Virol 87.12 (June 2013): 6986-6999.
PMID
23596289
Source
pubmed
Published In
Journal of virology
Volume
87
Issue
12
Publish Date
2013
Start Page
6986
End Page
6999
DOI
10.1128/JVI.00528-13

Production of prostaglandin E₂ in response to infection with modified vaccinia Ankara virus.

Prostaglandin E₂ (PGE₂) is an arachidonic acid (AA)-derived signaling molecule that can influence host immune responses to infection or vaccination. In this study, we investigated PGE₂ production in vitro by cells infected with the poxvirus vaccine strain, modified vaccinia Ankara virus (MVA). Human THP-1 cells, murine bone marrow-derived dendritic cells, and murine C3HA fibroblasts all accumulated PGE₂ to high levels in culture supernatants upon infection with MVA. We also demonstrated that MVA induced the release of AA from infected cells, and this was, most unusually, independent of host cytosolic phospholipase A₂ activity. The accumulation of AA and PGE₂ was dependent on viral gene expression, but independent of canonical NF-κB signaling via p65/RelA. The production of PGE₂ required host cyclooxygenase-2 (COX-2) activity, and COX-2 protein accumulated during MVA infection. The results of this study provide insight into a novel aspect of MVA biology that may affect the efficacy of MVA-based vaccines.

Authors
Pollara, JJ; Spesock, AH; Pickup, DJ; Laster, SM; Petty, ITD
MLA Citation
Pollara, JJ, Spesock, AH, Pickup, DJ, Laster, SM, and Petty, ITD. "Production of prostaglandin E₂ in response to infection with modified vaccinia Ankara virus." Virology 428.2 (July 2012): 146-155.
PMID
22534090
Source
epmc
Published In
Virology
Volume
428
Issue
2
Publish Date
2012
Start Page
146
End Page
155
DOI
10.1016/j.virol.2012.03.019

Cowpox virus induces interleukin-10 both in vitro and in vivo.

Cowpox virus infection induces interleukin-10 (IL-10) production from mouse bone marrow-derived dendritic cells (BMDCs) or cells of the mouse macrophage line (RAW264.7) at about 1800 pg/ml, whereas infections with vaccinia virus (strains WR or MVA) induced much less IL-10. Similarly, in vivo, IL-10 levels in bronchoalveolar lavage fluids of mice infected with cowpox virus were significantly higher than those after vaccinia virus infection. However, after intranasal cowpox virus infection, although dendritic and T-cell accumulations in the lungs of IL-10 deficient mice were greater than those in wild-type mice, weight-loss and viral burdens were not significantly different. IL-10 deficient mice were more susceptible than wild-type mice to re-infection with cowpox virus even though titers of neutralizing antibodies and virus-specific CD8 T cells were similar between IL-10 deficient and wild-type mice. Greater bronchopneumonia in IL-10 deficient mice than wild-type mice suggests that IL-10 contributes to the suppression of immunopathology in the lungs.

Authors
Spesock, AH; Barefoot, BE; Ray, CA; Kenan, DJ; Gunn, MD; Ramsburg, EA; Pickup, DJ
MLA Citation
Spesock, AH, Barefoot, BE, Ray, CA, Kenan, DJ, Gunn, MD, Ramsburg, EA, and Pickup, DJ. "Cowpox virus induces interleukin-10 both in vitro and in vivo." Virology 417.1 (August 15, 2011): 87-97.
PMID
21658738
Source
pubmed
Published In
Virology
Volume
417
Issue
1
Publish Date
2011
Start Page
87
End Page
97
DOI
10.1016/j.virol.2011.05.010

Cowpox virus inhibits human dendritic cell immune function by nonlethal, nonproductive infection.

Orthopoxviruses encode multiple proteins that modulate host immune responses. We determined whether cowpox virus (CPXV), a representative orthopoxvirus, modulated innate and acquired immune functions of human primary myeloid DCs and plasmacytoid DCs and monocyte-derived DCs (MDDCs). A CPXV infection of DCs at a multiplicity of infection of 10 was nonproductive, altered cellular morphology, and failed to reduce cell viability. A CPXV infection of DCs did not stimulate cytokine or chemokine secretion directly, but suppressed toll-like receptor (TLR) agonist-induced cytokine secretion and a DC-stimulated mixed leukocyte reaction (MLR). LPS-stimulated NF-κB nuclear translocation and host cytokine gene transcription were suppressed in CPXV-infected MDDCs. Early viral immunomodulatory genes were upregulated in MDDCs, consistent with early DC immunosuppression via synthesis of intracellular viral proteins. We conclude that a nonproductive CPXV infection suppressed DC immune function by synthesizing early intracellular viral proteins that suppressed DC signaling pathways.

Authors
Hansen, SJ; Rushton, J; Dekonenko, A; Chand, HS; Olson, GK; Hutt, JA; Pickup, D; Lyons, CR; Lipscomb, MF
MLA Citation
Hansen, SJ, Rushton, J, Dekonenko, A, Chand, HS, Olson, GK, Hutt, JA, Pickup, D, Lyons, CR, and Lipscomb, MF. "Cowpox virus inhibits human dendritic cell immune function by nonlethal, nonproductive infection." Virology 412.2 (April 10, 2011): 411-425.
PMID
21334039
Source
pubmed
Published In
Virology
Volume
412
Issue
2
Publish Date
2011
Start Page
411
End Page
425
DOI
10.1016/j.virol.2011.01.024

Cell biology. Propelling progeny.

Authors
Pickup, DJ
MLA Citation
Pickup, DJ. "Cell biology. Propelling progeny." Science 327.5967 (February 12, 2010): 787-788.
PMID
20150470
Source
pubmed
Published In
Science
Volume
327
Issue
5967
Publish Date
2010
Start Page
787
End Page
788
DOI
10.1126/science.1187160

Two mechanistically distinct immune evasion proteins of cowpox virus combine to avoid antiviral CD8 T cells.

Downregulation of MHC class I on the cell surface is an immune evasion mechanism shared by many DNA viruses, including cowpox virus. Previously, a cowpox virus protein, CPXV203, was shown to downregulate MHC class I. Here we report that CPXV12 is the only other MHC class I-regulating protein of cowpox virus and that it uses a mechanism distinct from that of CPXV203. Whereas CPXV203 retains fully assembled MHC class I by exploiting the KDEL-mediated endoplasmic reticulum retention pathway, CPXV12 binds to the peptide-loading complex and inhibits peptide loading on MHC class I molecules. Viruses deleted of both CPXV12 and CPXV203 demonstrated attenuated virulence in a CD8 T cell-dependent manner. These data demonstrate that CPXV12 and CPXV203 proteins combine to ablate MHC class I expression and abrogate antiviral CD8 T cell responses.

Authors
Byun, M; Verweij, MC; Pickup, DJ; Wiertz, EJHJ; Hansen, TH; Yokoyama, WM
MLA Citation
Byun, M, Verweij, MC, Pickup, DJ, Wiertz, EJHJ, Hansen, TH, and Yokoyama, WM. "Two mechanistically distinct immune evasion proteins of cowpox virus combine to avoid antiviral CD8 T cells." Cell Host Microbe 6.5 (November 19, 2009): 422-432.
PMID
19917497
Source
pubmed
Published In
Cell Host and Microbe
Volume
6
Issue
5
Publish Date
2009
Start Page
422
End Page
432
DOI
10.1016/j.chom.2009.09.012

Modified vaccinia virus Ankara can activate NF-kappaB transcription factors through a double-stranded RNA-activated protein kinase (PKR)-dependent pathway during the early phase of virus replication.

Modified vaccinia virus Ankara (MVA), which is a promising replication-defective vaccine vector, is unusual among the orthopoxviruses in activating NF-kappaB transcription factors in cells of several types. In human embryonic kidney (HEK 293T) cells, the MVA-induced depletion of IkappaBalpha required to activate NF-kappaB is inhibited by UV-inactivation of the virus, and begins before viral DNA replication. In HEK 293T, CHO, or RK13 cells, expression of the cowpox virus CP77 early gene, or the vaccinia virus K1L early gene suppresses MVA-induced IkappaBalpha depletion. In mouse embryonic fibroblasts (MEFs), MVA induction of IkappaBalpha depletion is dependent on the expression of mouse or human double-stranded RNA-activated protein kinase (PKR). These results demonstrate that events during the early phase of MVA replication can induce PKR-mediated processes contributing both to the activation of NF-kappaB signaling, and to processes that may restrict viral replication. This property may contribute to the efficacy of this vaccine virus.

Authors
Lynch, HE; Ray, CA; Oie, KL; Pollara, JJ; Petty, ITD; Sadler, AJ; Williams, BRG; Pickup, DJ
MLA Citation
Lynch, HE, Ray, CA, Oie, KL, Pollara, JJ, Petty, ITD, Sadler, AJ, Williams, BRG, and Pickup, DJ. "Modified vaccinia virus Ankara can activate NF-kappaB transcription factors through a double-stranded RNA-activated protein kinase (PKR)-dependent pathway during the early phase of virus replication." Virology 391.2 (September 2009): 177-186.
PMID
19596385
Source
epmc
Published In
Virology
Volume
391
Issue
2
Publish Date
2009
Start Page
177
End Page
186
DOI
10.1016/j.virol.2009.06.012

Vaccination with Venezuelan equine encephalitis replicons encoding cowpox virus structural proteins protects mice from intranasal cowpox virus challenge.

An anti-poxvirus vaccine based on replicon particles of Venezuelan equine encephalitis virus (VRP) is being developed. The cowpox virus genes encoding structural proteins corresponding to vaccinia virus proteins A33, B5, and A27 were each expressed from VRP. High serum IgG titers against these proteins were generated in BALB/c mice vaccinated with each of these VRP. VRP induced both IgG1 and IgG2a with a strong predominance of IgG2a production. The response is long-lasting, as evidenced by the retention of high anti-B5 serum IgG titers through at least 50 weeks after priming immunization. Mice vaccinated with B5-, A33- or A27-VRP individually or together survived intranasal challenge with cowpox virus, with the multivalent vaccine formulation providing more effective protection from weight loss and clinical signs of illness than the monovalent vaccines. These results demonstrate that VRP may provide an effective alternative to vaccinia virus vaccines against poxvirus infection.

Authors
Thornburg, NJ; Ray, CA; Collier, ML; Liao, H-X; Pickup, DJ; Johnston, RE
MLA Citation
Thornburg, NJ, Ray, CA, Collier, ML, Liao, H-X, Pickup, DJ, and Johnston, RE. "Vaccination with Venezuelan equine encephalitis replicons encoding cowpox virus structural proteins protects mice from intranasal cowpox virus challenge." Virology 362.2 (June 5, 2007): 441-452.
PMID
17292434
Source
pubmed
Published In
Virology
Volume
362
Issue
2
Publish Date
2007
Start Page
441
End Page
452
DOI
10.1016/j.virol.2007.01.001

Understanding orthopoxvirus interference with host immune responses to inform novel vaccine design.

Jenner's original vaccine used cowpox virus. Cowpox virus and, subsequently, vaccinia virus, a closely related Orthopoxvirus, provided the means to eradicate smallpox. This history and the unique properties of the virus suggest that vaccinia virus will continue to provide a useful vaccine platform. Yet, surprisingly, it has become apparent that much of the virus genome encodes accessory proteins that interfere with host immune responses to infection. Manipulation of these genes offers the potential for new generations of orthopoxvirus vaccines in which we will have far greater control over key features of the vaccination, including the sites of virus infection, the degree of virus replication, the pathogenicity of the virus and, most importantly, the suppression or induction of immune responses of specific types.

Authors
Pickup, DJ
MLA Citation
Pickup, DJ. "Understanding orthopoxvirus interference with host immune responses to inform novel vaccine design." Expert Rev Vaccines 6.1 (February 2007): 87-95. (Review)
PMID
17280481
Source
pubmed
Published In
Expert Review of Vaccines
Volume
6
Issue
1
Publish Date
2007
Start Page
87
End Page
95
DOI
10.1586/14760584.6.1.87

Post-transcription cleavage generates the 3' end of F17R transcripts in vaccinia virus.

Most vaccinia virus intermediate and late mRNAs possess 3' ends that are extremely heterogeneous in sequence. However, late mRNAs encoding the cowpox A-type inclusion protein (ATI), the second largest subunit of the RNA polymerase, and the late telomeric transcripts possess homogeneous 3' ends. In the case of the ATI mRNA, it has been shown that the homogeneous 3' end is generated by a post-transcriptional endoribonucleolytic cleavage event. We have determined that the F17R gene also produces homogeneous transcripts generated by a post-transcriptional cleavage event. Mapping of in vivo mRNA shows that the major 3' end of the F17R transcript maps 1262 nt downstream of the F17R translational start site. In vitro transcripts spanning the in vivo 3' end are cleaved in an in vitro reaction using extracts from virus infected cells, and the site of cleavage is the same both in vivo and in vitro. Cleavage is not observed using extract from cells infected in the presence of hydroxyurea; therefore, the cleavage factor is either virus-coded or virus-induced during the post-replicative phase of virus replication. The cis-acting sequence responsible for cleavage is orientation specific and the factor responsible for cleavage activity has biochemical properties similar to the factor required for cleavage of ATI transcripts. Partially purified cleavage factor generates cleavage products of expected size when either the ATI or F17R substrates are used in vitro, strongly suggesting that cleavage of both transcripts is mediated by the same factor.

Authors
D'Costa, SM; Antczak, JB; Pickup, DJ; Condit, RC
MLA Citation
D'Costa, SM, Antczak, JB, Pickup, DJ, and Condit, RC. "Post-transcription cleavage generates the 3' end of F17R transcripts in vaccinia virus." Virology 319.1 (February 5, 2004): 1-11.
PMID
14967483
Source
pubmed
Published In
Virology
Volume
319
Issue
1
Publish Date
2004
Start Page
1
End Page
11
DOI
10.1016/j.virol.2003.09.041

Identification of the orthopoxvirus p4c gene, which encodes a structural protein that directs intracellular mature virus particles into A-type inclusions.

The orthopoxvirus gene p4c has been identified in the genome of the vaccinia virus strain Western Reserve. This gene encodes the 58-kDa structural protein P4c present on the surfaces of the intracellular mature virus (IMV) particles. The gene is disrupted in the genome of cowpox virus Brighton Red (BR), demonstrating that although the P4c protein may be advantageous for virus replication in vivo, it is not essential for virus replication in vitro. Complementation and recombination analyses with the p4c gene have shown that the P4c protein is required to direct the IMV into the A-type inclusions (ATIs) produced by cowpox virus BR. The p4c gene is highly conserved among most members of the orthopoxvirus genus, including viruses that produce ATIs, such as cowpox, ectromelia, and raccoonpox viruses, as well as those such as variola, monkeypox, vaccinia, and camelpox viruses, which do not. The conservation of the p4c gene among the orthopoxviruses, irrespective of their capacities to produce ATIs, suggests that the P4c protein provides functions in addition to that of directing IMV into ATIs. These findings, and the presence of the P4c protein in IMV but not extracellular enveloped virus (D. Ulaeto, D. Grosenbach, and D. E. Hruby, J. Virol. 70:3372-3377, 1996), suggest a model in which the P4c protein may play a role in the retrograde movement of IMV particles, thereby contributing to the retention of IMV particles within the cytoplasm and within ATIs when they are present. In this way, the P4c protein may affect both viral morphogenesis and processes of virus dissemination.

Authors
McKelvey, TA; Andrews, SC; Miller, SE; Ray, CA; Pickup, DJ
MLA Citation
McKelvey, TA, Andrews, SC, Miller, SE, Ray, CA, and Pickup, DJ. "Identification of the orthopoxvirus p4c gene, which encodes a structural protein that directs intracellular mature virus particles into A-type inclusions." J Virol 76.22 (November 2002): 11216-11225.
PMID
12388681
Source
pubmed
Published In
Journal of virology
Volume
76
Issue
22
Publish Date
2002
Start Page
11216
End Page
11225

Cowpox virus encodes a fifth member of the tumor necrosis factor receptor family: a soluble, secreted CD30 homologue.

Cowpox virus (Brighton Red strain) possesses one of the largest genomes in the Orthopoxvirus genus. Sequence analysis of a region of the genome that is type-specific for cowpox virus identified a gene, vCD30, encoding a soluble, secreted protein that is the fifth member of the tumor necrosis factor receptor family known to be encoded by cowpox virus. The vCD30 protein contains 110 aa, including a 21-residue signal peptide, a potential O-linked glycosylation site, and a 58-aa sequence sharing 51-59% identity with highly conserved extracellular segments of both mouse and human CD30. A vCD30Fc fusion protein binds CD153 (CD30 ligand) specifically, and it completely inhibits CD153/CD30 interactions. Although the functions of CD30 are not well understood, the existence of vCD30 suggests that the cellular receptor plays a significant role in normal immune responses. Viral inhibition of CD30 also lends support to the potential therapeutic value of targeting CD30 in human inflammatory and autoimmune diseases.

Authors
Panus, JF; Smith, CA; Ray, CA; Smith, TD; Patel, DD; Pickup, DJ
MLA Citation
Panus, JF, Smith, CA, Ray, CA, Smith, TD, Patel, DD, and Pickup, DJ. "Cowpox virus encodes a fifth member of the tumor necrosis factor receptor family: a soluble, secreted CD30 homologue." Proc Natl Acad Sci U S A 99.12 (June 11, 2002): 8348-8353.
PMID
12034885
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
99
Issue
12
Publish Date
2002
Start Page
8348
End Page
8353
DOI
10.1073/pnas.122238599

Cowpox virus and other members of the orthopoxvirus genus interfere with the regulation of NF-kappaB activation.

NF-kappaB comprises a family of transcription factors that regulate key immune processes. In this study, the effects of orthopoxvirus infection upon the activation of NF-kappaB were examined. During the early phase of infection, cowpox virus can inhibit the induction of NF-kappaB-regulated gene expression by interfering with the process of IkappaBalpha degradation. Although either okadaic acid or tumor necrosis factor (TNF) treatment of infected cells can induce IkappaBalpha phosphorylation, further processing of IkappaBalpha is inhibited. These results suggest that cowpox virus is capable of inhibiting the activation of NF-kappaB at a point where multiple signal transduction pathways converge. Other orthopoxviruses affect NF-kappaB activity, but in a type-specific manner. Raccoonpox virus and vaccinia virus (Copenhagen strain) negatively affect NF-kappaB induction by TNF. In contrast, the modified vaccinia virus Ankara strain induces NF-kappaB activation, even in the absence of other stimuli. These findings suggest that orthopoxviruses may affect a broad range of virus-host interactions through their effects upon NF-kappaB activation. Moreover, because of the central role for NF-kappaB in immune processes and disease, these type-specific effects may contribute significantly to the immunogenic and pathogenic properties of poxviruses.

Authors
Oie, KL; Pickup, DJ
MLA Citation
Oie, KL, and Pickup, DJ. "Cowpox virus and other members of the orthopoxvirus genus interfere with the regulation of NF-kappaB activation." Virology 288.1 (September 15, 2001): 175-187.
PMID
11543670
Source
pubmed
Published In
Virology
Volume
288
Issue
1
Publish Date
2001
Start Page
175
End Page
187
DOI
10.1006/viro.2001.1090

A 43-nucleotide RNA cis-acting element governs the site-specific formation of the 3' end of a poxvirus late mRNA.

The 3' ends of late mRNAs of the ati gene, encoding the major component of the A-type inclusions, are generated by endoribonucleolytic cleavage at a specific site in the primary transcript [Antczak et al., (1992), Proc. Natl. Acad. Sci. USA 89, 12033-12037]. In this study, sequence analysis of cDNAs of the 3' ends of ati mRNAs showed these mRNAs are 3' polyadenylated at the RNA cleavage site. This suggests that ati mRNA 3' end formation involves cleavage of a late transcript, with subsequent 3' polyadenylation of the 5' cleavage product. The RNA cis-acting element, the AX element, directing orientation-dependent formation of these mRNA 3' ends, was mapped to a 345-bp AluI-XbaI fragment. Deletion analyses of this fragment showed that the boundaries of the AX element are within -5 and +38 of the RNA cleavage site. Scanning mutagenesis showed that the AX element contains at least two subelements: subelement I, 5'-UUUAU downward arrowCCGAUAAUUC-3', containing the cleavage site ( downward arrow), separated from the downstream subelement II, 5'-AAUUUCGGAUUUGAAUGC-3', by a 10-nucleotide region, whose composition may be altered without effect on RNA 3' end formation. These features, which differ from those of other elements controlling RNA processing, suggest that the AX element is a component of a novel mechanism of RNA 3' end formation.

Authors
Howard, ST; Ray, CA; Patel, DD; Antczak, JB; Pickup, DJ
MLA Citation
Howard, ST, Ray, CA, Patel, DD, Antczak, JB, and Pickup, DJ. "A 43-nucleotide RNA cis-acting element governs the site-specific formation of the 3' end of a poxvirus late mRNA." Virology 255.1 (March 1, 1999): 190-204.
PMID
10049834
Source
pubmed
Published In
Virology
Volume
255
Issue
1
Publish Date
1999
Start Page
190
End Page
204
DOI
10.1006/viro.1998.9547

A third distinct tumor necrosis factor receptor of orthopoxviruses.

Cowpox virus Brighton red strain (CPV) contains a gene, crmD, which encodes a 320-aa tumor necrosis factor receptor (TNFR) of 44% and 22% identity, respectively, to the CPV TNFR-like proteins, cytokine response modifiers (crm) CrmB and CrmC. The crmD gene was interrupted in three other cowpox strains examined and absent in various other orthopoxviruses; however, four strains of ectromelia virus (ECT) examined contained an intact crmD (97% identity to CPV crmD) and lacked cognates of crmB and crmC. The protein, CrmD, contains a transport signal; a 151-aa cysteine-rich region with 21 cysteines that align with human TNFRII ligand-binding region cysteines; and C-terminal region sequences that are highly diverged from cellular TNFR C-terminal region sequences involved in signal transduction. Bacterial maltose-binding proteins containing the CPV or ECT CrmD cysteine-rich region bound TNF and lymphotoxin-alpha (LTalpha) and blocked their in vitro cytolytic activity. Secreted viral CrmD bound TNF and LTalpha and was detectable after the early stage of replication, using nonreducing conditions, as 60- to 70-kDa predominant and 90- to 250-kDa minor disulfide-linked complexes that were able to be reduced to a 46-kDa form and deglycosylated to a 38-kDa protein. Cells infected with CPV produced extremely low amounts of CrmD compared with ECT. Possessing up to three TNFRs, including CrmD, which is secreted as disulfide-linked complexes in varied amounts by CPV and ECT, likely enhances the dynamics of the immune modulating mechanisms of orthopoxviruses.

Authors
Loparev, VN; Parsons, JM; Knight, JC; Panus, JF; Ray, CA; Buller, RM; Pickup, DJ; Esposito, JJ
MLA Citation
Loparev, VN, Parsons, JM, Knight, JC, Panus, JF, Ray, CA, Buller, RM, Pickup, DJ, and Esposito, JJ. "A third distinct tumor necrosis factor receptor of orthopoxviruses." Proc Natl Acad Sci U S A 95.7 (March 31, 1998): 3786-3791.
PMID
9520445
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
95
Issue
7
Publish Date
1998
Start Page
3786
End Page
3791

Poxvirus genomes encode a secreted, soluble protein that preferentially inhibits beta chemokine activity yet lacks sequence homology to known chemokine receptors.

Poxvirus genomes encode several proteins which inhibit specific elements of the host immune response. We show the "35K" virulence gene in variola and cowpox viruses, whose vaccinia and Shope fibroma virus equivalents are strongly conserved in sequence, actually encodes a secreted soluble protein with high-affinity binding to virtually all known beta chemokines, but only weak or no affinity to the alpha and gamma classes. The viral protein completely inhibits the biological activity of monocyte chemotactic protein-1 (MCP-1) by competitive inhibition of chemokine binding to cellular receptors. As all beta chemokines are also shown to cross-compete with MCP1 binding to the viral protein, we conclude that this viral chemokine inhibitor (vCCI) not only interacts through a common binding site, but is likely a potent general inhibitor of beta chemokine activity. Unlike many poxvirus virulence genes to date, which are clearly altered forms of acquired cellular genes of the vertebrate immune system, this viral chemokine inhibitor (vCCI) shares no sequence homology with known proteins, including known cellular chemokine receptors, all of which are multiple membrane-spanning proteins. Thus, vCCI presumably has no cellular analogue and instead may be the product of unrelenting sequence variations which gave rise to a completely new protein with similar binding properties to native chemokine receptors. The proposed function of vCCI is inhibition of the proinflammatory (antiviral) activities of beta chemokines.

Authors
Smith, CA; Smith, TD; Smolak, PJ; Friend, D; Hagen, H; Gerhart, M; Park, L; Pickup, DJ; Torrance, D; Mohler, K; Schooley, K; Goodwin, RG
MLA Citation
Smith, CA, Smith, TD, Smolak, PJ, Friend, D, Hagen, H, Gerhart, M, Park, L, Pickup, DJ, Torrance, D, Mohler, K, Schooley, K, and Goodwin, RG. "Poxvirus genomes encode a secreted, soluble protein that preferentially inhibits beta chemokine activity yet lacks sequence homology to known chemokine receptors." Virology 236.2 (September 29, 1997): 316-327.
PMID
9325239
Source
pubmed
Published In
Virology
Volume
236
Issue
2
Publish Date
1997
Start Page
316
End Page
327
DOI
10.1006/viro.1997.8730

Cowpox virus genome encodes a second soluble homologue of cellular TNF receptors, distinct from CrmB, that binds TNF but not LT alpha.

We show the cowpox genome (Brighton Red strain) contains a single copy gene, crmC, expressed at late times during viral infection, encoding a soluble, secreted protein whose sequence marks it as a new member of the TNF receptor family. The cysteine-rich protein contains 186 amino acids, the N-terminal 21 of which constitute a signal peptide, and two potential N-linked glycosylation sites. The approximately 25-kDa recombinant protein binds TNF specifically and completely inhibits TNF-mediated cytolysis. The strongest sequence homologues are the ligand-binding regions of the type II cellular TNF receptor (TNFRII) and CrmB, a distinct pox virus gene also encoding a soluble TNF binding protein. Unlike TNFRII and CrmB, CrmC does not bind lymphotoxin (LT alpha, TNF beta) and lacks the conserved (but nonhomologous) approximately 150-residue C-terminal domain of CrmB proteins. The presumed function of CrmC is viral inhibition of host-elicited TNF.

Authors
Smith, CA; Hu, FQ; Smith, TD; Richards, CL; Smolak, P; Goodwin, RG; Pickup, DJ
MLA Citation
Smith, CA, Hu, FQ, Smith, TD, Richards, CL, Smolak, P, Goodwin, RG, and Pickup, DJ. "Cowpox virus genome encodes a second soluble homologue of cellular TNF receptors, distinct from CrmB, that binds TNF but not LT alpha." Virology 223.1 (September 1, 1996): 132-147.
PMID
8806547
Source
pubmed
Published In
Virology
Volume
223
Issue
1
Publish Date
1996
Start Page
132
End Page
147
DOI
10.1006/viro.1996.0462

Proteolytic activation of the cell death protease Yama/CPP32 by granzyme B.

The serine protease granzyme B, which is secreted by cytotoxic cells, is one of the major effectors of apoptosis in susceptible targets. To examine the apoptotic mechanism of granzyme B, we have analyzed its effect on purified proteins that are thought to be components of death pathways inherent to cells. We demonstrate that granzyme B processes interleukin 1beta-converting enzyme (ICE) and the ICE-related protease Yama (also known as CPP32 or apopain) by limited proteolysis. Processing of ICE does not lead to activation. However, processing by granzyme B leads directly to the activation of Yama, which is now able to bind inhibitors and cleave the substrate poly(ADP-ribose) polymerase whose proteolysis is a marker of apoptosis initiated by several other stimuli. Thus ICE-related proteases can be activated by serine proteases that possess the correct specificity. Activation of pro-Yama by granzyme B is within the physiologic range. Thus the cytotoxic effect of granzyme B can be explained by its activation of an endogenous protease component of a programmed cell death pathway.

Authors
Quan, LT; Tewari, M; O'Rourke, K; Dixit, V; Snipas, SJ; Poirier, GG; Ray, C; Pickup, DJ; Salvesen, GS
MLA Citation
Quan, LT, Tewari, M, O'Rourke, K, Dixit, V, Snipas, SJ, Poirier, GG, Ray, C, Pickup, DJ, and Salvesen, GS. "Proteolytic activation of the cell death protease Yama/CPP32 by granzyme B." Proc Natl Acad Sci U S A 93.5 (March 5, 1996): 1972-1976.
PMID
8700869
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
93
Issue
5
Publish Date
1996
Start Page
1972
End Page
1976

The mode of death of pig kidney cells infected with cowpox virus is governed by the expression of the crmA gene.

Pig kidney cells (LLC-PK1) were infected with one of three viruses: wild-type cowpox virus (Brighton red strain) expressing the crmA gene; recombinant cowpox virus A602, lacking the crmA gene; or cowpox virus A604, a revertant of virus A602, expressing the crmA gene. The wild-type virus and virus A604 produced identical cytopathic effects consistent with death by necrosis. In these cells, the structural features of the plasma membrane, the nuclear membrane, and the chromatin were maintained until lysis of the cells. In contrast, cowpox virus A602 produced cytopathic effects consistent with death by apoptosis. These effects included loss of microvilli on the cell surface, margination and condensation of the chromatin, progressive convolution of the nuclear membrane, release of dense chromatin masses on disintegration of the nucleus, fragmentation of the DNA, and the generation of apoptotic bodies. These results suggest that the crmA gene is necessary to inhibit processes of apoptosis induced in LLC-PK1 cells by infection with cowpox virus. Thus in cells of certain types, the crmA gene can act with other viral genes to control the mode of death of the virus-infected cell. This capability may be advantageous to virus replication in vivo, potentially facilitating both virus trafficking and interference with antiviral immune defenses.

Authors
Ray, CA; Pickup, DJ
MLA Citation
Ray, CA, and Pickup, DJ. "The mode of death of pig kidney cells infected with cowpox virus is governed by the expression of the crmA gene." Virology 217.1 (March 1, 1996): 384-391.
PMID
8599227
Source
pubmed
Published In
Virology
Volume
217
Issue
1
Publish Date
1996
Start Page
384
End Page
391
DOI
10.1006/viro.1996.0128

Granzyme B is inhibited by the cowpox virus serpin cytokine response modifier A.

The ability of cytolytic cells to cause apoptosis in target cells is in part due to the action of the serine proteinase granzyme B. We demonstrate that granzyme B is inhibited, with an association rate constant of 2.9 x 10(5) M-1 s-1, by the cowpox viral serpin cytokine response modifier A (CrmA). Previously we have shown CrmA to be an inhibitor of the cysteine proteinase interleukin-1 beta-converting enzyme (ICE). Thus the mechanism of CrmA involves the unusual ability to efficiently inhibit proteinases from two distinct catalytic classes, in this case serine and cysteine proteinases. Granzyme B and ICE are both used to combat viral infection, and we propose that cowpox virus uses CrmA to evade the contribution of these two proteinases. Thus, through CrmA, the virus may influence two of the pathways normally used to kill virus-infected cells: acting on endogenous proteinases such as ICE and on exogenous proteinases delivered by cytotoxic lymphocytes to infected cells.

Authors
Quan, LT; Caputo, A; Bleackley, RC; Pickup, DJ; Salvesen, GS
MLA Citation
Quan, LT, Caputo, A, Bleackley, RC, Pickup, DJ, and Salvesen, GS. "Granzyme B is inhibited by the cowpox virus serpin cytokine response modifier A." J Biol Chem 270.18 (May 5, 1995): 10377-10379.
PMID
7737968
Source
pubmed
Published In
The Journal of biological chemistry
Volume
270
Issue
18
Publish Date
1995
Start Page
10377
End Page
10379

Human IL-1 beta processing and secretion in recombinant baculovirus-infected Sf9 cells is blocked by the cowpox virus serpin crmA.

Biologically active, mature IL-1 beta (mIL-1 beta) is released from activated monocytes after proteolytic processing from an inactive precursor (pIL-1 beta). IL-1 beta converting enzyme (ICE), the first member of a newly discovered family of cysteine proteinases, is required for this processing event. The cleaved cytokine is released from monocytes by an unknown mechanism which does not employ a standard hydrophobic signal sequence. As in mammalian fibroblasts, insect Sf9 cells do not normally process or secrete human IL-1 beta. The expression of active ICE enables Sf9 cells to process 31-kDa pIL-1 beta correctly at Asp27 and Asp116, and to export 17.5-kDa mIL-1 beta. The recombinant heterodimeric human enzyme purified from Sf9 cells possesses a sp. act. of 2.9 +/- 0.5 x 10(6) U/mg and is indistinguishable from native ICE with regard to its subunit composition and catalytic properties. In this system, co-expression of the cowpox virus crmA gene, an extremely potent serpin inhibitor of ICE (Ki < 7 pM), inhibits ICE activation completely and blocks pIL-1 beta processing and mIL-1 beta secretion by approximately 95%. The results indicate that ICE, in addition to its processing function, facilitates the transport of IL-1 beta across the plasma membrane.

Authors
Howard, AD; Palyha, OC; Griffin, PR; Peterson, EP; Lenny, AB; Ding, GJ; Pickup, DJ; Thornberry, NA; Schmidt, JA; Tocci, MJ
MLA Citation
Howard, AD, Palyha, OC, Griffin, PR, Peterson, EP, Lenny, AB, Ding, GJ, Pickup, DJ, Thornberry, NA, Schmidt, JA, and Tocci, MJ. "Human IL-1 beta processing and secretion in recombinant baculovirus-infected Sf9 cells is blocked by the cowpox virus serpin crmA." J Immunol 154.5 (March 1, 1995): 2321-2332.
PMID
7868902
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
154
Issue
5
Publish Date
1995
Start Page
2321
End Page
2332

Cowpox virus contains two copies of an early gene encoding a soluble secreted form of the type II TNF receptor.

The inverted terminal repeats of the DNA of cowpox virus (Brighton Red strain) contain the crmB gene, an additional member of a family of viral genes that modify cytokine responses to infection. The crmB gene is transcribed from an early promoter. The primary product is a 355-amino-acid protein containing a signal peptide sequence and three potential N-linked glycosylation sites. The mature gene product is a secreted soluble protein that has an apparent molecular mass of 48 kDa. TNF alpha and TNF beta bind to this protein in a competitive manner, consistent with the sequence of its N-terminal 176 amino acids, which closely resembles the ligand-binding domains of the type II (75-kDa) human TNF receptor. The sequence of the C-terminal 161 amino acids of the CrmB protein is unlike that of human TNF receptors, but overall, the CrmB protein is similar to the T2 proteins of the leporipoxviruses (48% identity) and the predicted product of the G4R/G2R open reading frame of variola virus (85% identity), suggesting that not only the TNF-binding domains but also the C-terminal regions contribute to the functions of these viral proteins. These results show that orthopoxiviruses such as cowpox virus encode secreted forms of TNF receptors that can contribute to the modification of TNF-mediated antiviral processes.

Authors
Hu, FQ; Smith, CA; Pickup, DJ
MLA Citation
Hu, FQ, Smith, CA, and Pickup, DJ. "Cowpox virus contains two copies of an early gene encoding a soluble secreted form of the type II TNF receptor." Virology 204.1 (October 1994): 343-356.
PMID
8091665
Source
pubmed
Published In
Virology
Volume
204
Issue
1
Publish Date
1994
Start Page
343
End Page
356
DOI
10.1006/viro.1994.1539

Inhibition of interleukin-1 beta converting enzyme by the cowpox virus serpin CrmA. An example of cross-class inhibition.

We reported previously that human interleukin-1 beta converting enzyme (ICE) is regulated by the CrmA serpin encoded by cowpox virus. We now report the mechanism and kinetics of this unusual inhibition of a cysteine proteinase by a member of the serpin superfamily previously thought to inhibit serine proteinase only. CrmA possesses several characteristics typical of a number of inhibitory serpins. It is conformationally unstable, unfolding around 3 M urea, and stable to denaturation in 8 M urea upon complex formation with ICE. CrmA rapidly inhibits ICE with an association rate constant (kon) of 1.7 x 10(7) M-1 s-1, forming a tight complex with an equilibrium constant for inhibition (Ki) of less than 4 x 10(-12) M. These data indicate that CrmA is a potent inhibitor of ICE, consistent with the dramatic effects of CrmA on modifying host responses to virus infection. The inhibition of ICE by CrmA is an example of a "cross-class" interaction, in which a serpin inhibits a non-serine proteinase. Since CrmA possesses characteristics shared by inhibitors of serine proteinases, we presume that ICE, though it is a cysteine proteinase, has a substrate binding geometry strikingly close to that of serine proteinases. We reason that it is the substrate binding geometry, not the catalytic mechanism of a proteinase, that dictates its reactivity with protein inhibitors.

Authors
Komiyama, T; Ray, CA; Pickup, DJ; Howard, AD; Thornberry, NA; Peterson, EP; Salvesen, G
MLA Citation
Komiyama, T, Ray, CA, Pickup, DJ, Howard, AD, Thornberry, NA, Peterson, EP, and Salvesen, G. "Inhibition of interleukin-1 beta converting enzyme by the cowpox virus serpin CrmA. An example of cross-class inhibition." J Biol Chem 269.30 (July 29, 1994): 19331-19337.
PMID
8034697
Source
pubmed
Published In
The Journal of biological chemistry
Volume
269
Issue
30
Publish Date
1994
Start Page
19331
End Page
19337

Poxviral modifiers of cytokine responses to infection.

Poxviruses include some of the most virulent of all human pathogens. In part, the virulence of these viruses stems from their abilities to counter host defenses against infection. A family of cytokine-response modifiers encoded by the poxviruses contribute to these countermeasures. The poxviral cytokine-response modifiers appear to affect cytokine responses in at least four different ways: (a) by inhibiting the synthesis and release of cytokines from infected cells; (b) by interfering with the interaction between a cytokine and its receptor; (c) by inhibiting cytokine signal transmission; and (d) by synthesizing virus-encoded cytokines that antagonize the effects of host cytokines mediating antiviral processes. Known poxviral, cytokine-response modifiers include CrmA, an inhibitor of the interleukin-1 beta converting enzyme; several secreted soluble receptors for tumor necrosis factor, interleukin-1, and interferon-gamma; and poxvirus-encoded growth factors resembling epidermal growth factor. Collectively, these and other as yet unidentified cytokine-response modifiers contribute to the inhibition of a variety of nonspecific and virus-specific immune defenses against virus infection. Information gained on the mechanisms used by poxviruses to modify cytokine-mediated processes should assist the development of novel therapies for a variety of diseases.

Authors
Pickup, DJ
MLA Citation
Pickup, DJ. "Poxviral modifiers of cytokine responses to infection." Infect Agents Dis 3.2-3 (April 1994): 116-127. (Review)
PMID
7812650
Source
pubmed
Published In
Infectious Agents and Disease
Volume
3
Issue
2-3
Publish Date
1994
Start Page
116
End Page
127

In vitro expression of serpins.

Authors
Komiyama, T.,, T; Quan, L; Snipas,, S; Ray, CA; Pickup, DJ; Salvesen, G
MLA Citation
Komiyama, T., T, Quan, L, Snipas, S, Ray, CA, Pickup, DJ, and Salvesen, G. "In vitro expression of serpins." Techniques in Protein Chemistry V. Ed. JW Crabb. New York: Academic Press, 1994. 305-312. (Chapter)
Source
manual
Publish Date
1994
Start Page
305
End Page
312

IN-VITRO EXPRESSION OF SERPINS

Authors
KOMIYAMA, T; QUAN, L; SNIPAS, S; RAY, CA; PICKUP, DJ; SALVESEN, G
MLA Citation
KOMIYAMA, T, QUAN, L, SNIPAS, S, RAY, CA, PICKUP, DJ, and SALVESEN, G. "IN-VITRO EXPRESSION OF SERPINS." 1994.
Source
wos-lite
Published In
TECHNIQUES IN PROTEIN CHEMISTRY V
Publish Date
1994
Start Page
305
End Page
312

SOLUBLE TUMOR-NECROSIS-FACTOR RECEPTORS OF 2 TYPES ARE ENCODED BY COWPOX VIRUS

Authors
PICKUP, DJ; HU, FQ; GOODWIN, RG; DAVIS, T; SMITH, C
MLA Citation
PICKUP, DJ, HU, FQ, GOODWIN, RG, DAVIS, T, and SMITH, C. "SOLUBLE TUMOR-NECROSIS-FACTOR RECEPTORS OF 2 TYPES ARE ENCODED BY COWPOX VIRUS." January 26, 1993.
Source
wos-lite
Published In
Journal of Cellular Biochemistry
Publish Date
1993
Start Page
81
End Page
81

Site-specific RNA cleavage generates the 3' end of a poxvirus late mRNA.

The cowpox virus late mRNAs encoding the major protein of the A-type inclusions have 3' ends corresponding to a single site in the DNA template. The DNA sequence of the Alu I-Xba I fragment at this position encodes an RNA cis-acting signal, designated the AX element, which directs this RNA 3' end formation. In cells infected with vaccinia virus the AX element functions independently of either the nature of the promoter element or the RNA polymerase responsible for generating the primary RNA. At late times during virus replication, vaccinia virus induces or activates a site-specific endoribonuclease that cleaves primary RNAs within the AX element. The 3' end produced by RNA cleavage is then polyadenylylated to form the 3' end of the mature mRNA. Therefore, the poxviruses employ at least two mechanisms of RNA 3' end formation during the viral replication cycle. One mechanism, which is operative at early times in viral replication, involves the termination of transcription [Rohrmann, G., Yuen, L. & Moss, B. (1986) Cell 46, 1029-1035]. A second mechanism, which is operative at late times during viral replication, involves the site-specific cleavage of primary RNAs.

Authors
Antczak, JB; Patel, DD; Ray, CA; Ink, BS; Pickup, DJ
MLA Citation
Antczak, JB, Patel, DD, Ray, CA, Ink, BS, and Pickup, DJ. "Site-specific RNA cleavage generates the 3' end of a poxvirus late mRNA." Proc Natl Acad Sci U S A 89.24 (December 15, 1992): 12033-12037.
PMID
1465436
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
89
Issue
24
Publish Date
1992
Start Page
12033
End Page
12037

Vaccinia and cowpox viruses encode a novel secreted interleukin-1-binding protein.

Supernatants from vaccinia virus (VV)-infected CV-1 cells were examined and found to contain a 33 kd protein capable of binding murine interleukin-1 beta (mIL-1 beta). A VV open reading frame (ORF) that exhibits 30% amino acid identity to the type II IL-1 receptor was expressed in CV-1-EBNA cells and shown specifically to bind mIL-1 beta. A similar ORF from cowpox virus was expressed and also specifically bound mIL-1 beta. A recombinant VV was constructed in which this ORF was disrupted (vB15RKO). Supernatants from vB15RKO-infected cells did not contain an IL-1-binding protein. Supernatants from VV-infected CV-1 cells were capable of inhibiting IL-1-induced murine lymphocyte proliferation in vitro while supernatants from vB15RKO infected cells did not. Intracranial inoculation of mice with vB15RKO suggests that this ORF is involved in VV virulence. The possible role of a virus-encoded IL-1-binding protein in the pathology of a poxvirus infection and its relationship to other poxvirus-encoded immune modulators is discussed.

Authors
Spriggs, MK; Hruby, DE; Maliszewski, CR; Pickup, DJ; Sims, JE; Buller, RM; VanSlyke, J
MLA Citation
Spriggs, MK, Hruby, DE, Maliszewski, CR, Pickup, DJ, Sims, JE, Buller, RM, and VanSlyke, J. "Vaccinia and cowpox viruses encode a novel secreted interleukin-1-binding protein." Cell 71.1 (October 2, 1992): 145-152.
PMID
1339315
Source
pubmed
Published In
Cell
Volume
71
Issue
1
Publish Date
1992
Start Page
145
End Page
152

The eukaryotic translation initiation factor 4E is not modified during the course of vaccinia virus replication.

The ability of vaccinia virus to inhibit processes of cap-dependent translational initiation by inactivating the eukaryotic translation initiation factor 4E (eIF-4E) has been examined. Analyses of the quantities of eIF-4E present in either uninfected mouse L929 cells or vaccinia virus-infected cells showed that during the first 12 hr of virus replication, when there is a marked decrease in host gene expression in infected cells, there is no change in the total amount of eIF-4E present. Analyses of eIF-4E that was metabolically labeled with [32P] and then purified by affinity chromatography using m7GTP-Sepharose 4B, indicated that neither the incorporation of radiolabel into eIF-4E nor the amounts of eIF-4E capable of binding to cap structures changed significantly during virus replication. Immunodetection of phosphorylated and unphosphorylated eIF-4E in cell lysates fractionated by two-dimensional gel electrophoresis showed that the steady-state levels of phosphorylated and unphosphorylated forms of eIF-4E were similar in uninfected and virus-infected cells. These results suggest that vaccinia virus does not gain preferential translation of viral mRNAs over other mRNAs in the cell by reducing either eIF-4E phosphorylation or its ability to bind to the cap structure.

Authors
Gierman, TM; Frederickson, RM; Sonenberg, N; Pickup, DJ
MLA Citation
Gierman, TM, Frederickson, RM, Sonenberg, N, and Pickup, DJ. "The eukaryotic translation initiation factor 4E is not modified during the course of vaccinia virus replication." Virology 188.2 (June 1992): 934-937.
PMID
1585661
Source
pubmed
Published In
Virology
Volume
188
Issue
2
Publish Date
1992
Start Page
934
End Page
937

Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1 beta converting enzyme.

Cowpox virus effectively inhibits inflammatory responses against viral infection in the chick embryo. This study demonstrates that one of the viral genes necessary for this inhibition, the crmA gene (a cytokine response modifier gene), encodes a serpin that is a specific inhibitor of the interleukin-1 beta converting enzyme. This serpin can prevent the proteolytic activation of interleukin-1 beta, thereby suppressing an interleukin-1 beta response to infection. However, the modification of this single cytokine response is not sufficient to inhibit inflammatory responses. This suggests that cowpox virus encodes several cytokine response modifiers that act together to inhibit the release of pro-inflammatory cytokines in response to infection. These viral countermeasures to host defenses against infection may contribute significantly to the pathology associated with poxvirus infections.

Authors
Ray, CA; Black, RA; Kronheim, SR; Greenstreet, TA; Sleath, PR; Salvesen, GS; Pickup, DJ
MLA Citation
Ray, CA, Black, RA, Kronheim, SR, Greenstreet, TA, Sleath, PR, Salvesen, GS, and Pickup, DJ. "Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1 beta converting enzyme." Cell 69.4 (May 15, 1992): 597-604.
PMID
1339309
Source
pubmed
Published In
Cell
Volume
69
Issue
4
Publish Date
1992
Start Page
597
End Page
604

Molecular cloning of the IL-1β processing enzyme.

Authors
Pickup, DJ
MLA Citation
Pickup, DJ. "Molecular cloning of the IL-1β processing enzyme." Pathophysiology and Pharmacology of Cytokines. Ed. P Ghezzi, and A Mantovani. Augusta, Georgia, USA: Biomedical Press, 1992. 3-8. (Essay)
Source
manual
Publish Date
1992
Start Page
3
End Page
8

Transcription of the terminal loop region of vaccinia virus DNA is initiated from the telomere sequences directing DNA resolution.

The telomeres of vaccinia virus DNA are transcribed at late times after infection. Analysis of cDNAs of RNA transcripts of the terminal loop region of the viral DNA shows that both inverted and complementary forms of the terminal loop region are transcribed. These late RNAs, which contain 5' poly(A) sequences, do not appear to encode any proteins. The transcriptional start sites for most of these RNAs are within the sequences that direct the resolution of concatemeric DNA replication intermediates (M. Merchlinsky and B. Moss, 1989, J. Virol. 63, 4354-4361). This suggests that the process of DNA resolution may involve transcription initiated from the telomere sequences required for resolution.

Authors
Hu, FQ; Pickup, DJ
MLA Citation
Hu, FQ, and Pickup, DJ. "Transcription of the terminal loop region of vaccinia virus DNA is initiated from the telomere sequences directing DNA resolution." Virology 181.2 (April 1991): 716-720.
PMID
2014645
Source
pubmed
Published In
Virology
Volume
181
Issue
2
Publish Date
1991
Start Page
716
End Page
720

Transcription of orthopoxvirus telomeres at late times during infection.

The telomeres of orthopoxvirus DNAs consists largely of short repeated sequences organized into at least two separate sets. Although the sequence composition of the orthopoxvirus telomeres is highly conserved, these regions do not appear to encode any proteins. At late times during infection, the telomeres of vaccinia virus are transcribed. A promoter in the region between the two sets of repeats directs transcription towards the hairpin-loop end of the viral DNA. This promoter resembles the promoters of other poxvirus late genes, and directs the synthesis of RNAs whose structure is consistent with the presence of 5' poly(A) sequences typical of late RNAs. The lengths of these late transcripts suggest that some transcription extends through the hairpin-loop region. This might occur either when the genome is in a monomeric form or when the genome is in the concatemeric form of the DNA replication intermediate. The function of late transcription of the telomeres is unclear, but similar transcription of the telomeres of vaccinia virus, cowpox virus, and raccoonpox virus suggests that such transcription may have a role in viral replication.

Authors
Parsons, BL; Pickup, DJ
MLA Citation
Parsons, BL, and Pickup, DJ. "Transcription of orthopoxvirus telomeres at late times during infection." Virology 175.1 (March 1990): 69-80.
PMID
2309453
Source
pubmed
Published In
Virology
Volume
175
Issue
1
Publish Date
1990
Start Page
69
End Page
80

Vaccinia virus directs the synthesis of early mRNAs containing 5' poly(A) sequences.

mRNAs transcribed from late promoters of several poxvirus genes contain 5' poly(A) sequences that are not complementary to the viral DNA. In contrast, early mRNAs containing 5' poly(A) sequences have not previously been identified. Modifications to the sequence of the promoter of an early gene of cowpox virus enable this promoter to direct the synthesis of RNAs containing 5' poly(A) sequences. When the sequence 3'-ATTTA-5', which is present at the RNA start-sites of several late promoters, is positioned such that the RNA start-site of the early promoter is at the first thymidylate in this sequence, this early promoter directs the synthesis of early RNAs containing 4-11 adenylates at their 5' ends. When two of the thymidylates in the sequence 3'-ATTTA-5' are removed, the promoter directs the synthesis of early RNAs lacking 5' poly(A) sequences. These results are consistent with the proposal that 5' polyadenylylation of poxvirus RNAs occurs by repetitive transcription of thymidylates in the sequence 3'-ATTTA-5' often present at the sites of transcriptional initiation. In addition, these results demonstrate that 5' polyadenylylation of viral RNAs is not exclusively a late function. The promoter regions of a few early genes of vaccinia virus contain the sequence 3'-ATTTA-5'. Analyses of the transcripts of one of these genes, the D5 gene, indicated that these mRNAs contain 5' poly(A) sequences, suggesting that early mRNAs of a subset of viral genes contain 5' poly(A) sequences.

Authors
Ink, BS; Pickup, DJ
MLA Citation
Ink, BS, and Pickup, DJ. "Vaccinia virus directs the synthesis of early mRNAs containing 5' poly(A) sequences." Proc Natl Acad Sci U S A 87.4 (February 1990): 1536-1540.
PMID
1968253
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
87
Issue
4
Publish Date
1990
Start Page
1536
End Page
1540

Fine structure mapping and phenotypic analysis of five temperature-sensitive mutations in the second largest subunit of vaccinia virus DNA-dependent RNA polymerase.

We have used plasmid clones spanning the region encoding the 132-kDa subunit of the cowpox virus RNA polymerase (CPV rpo 132) to marker rescue each of five vaccinia virus (VV) temperature sensitive (ts) mutants, ts 27, ts 29, ts 32, ts 47, and ts 62, which together constitute a single complementation group. The experiments fine-map the vaccinia mutations to a 1.3-kb region containing the 3' end of the CPV rpo 132 gene. Phenotypic characterization shows that all five mutants are affected to varying extents in their ability to synthesize late viral proteins at the nonpermissive temperature, similar to other ts mutants with lesions in the 22- and the 147-kDa subunits of the VV RNA polymerase. Two mutants, ts 27 and ts 32, exhibit a delay in the synthesis of late viral proteins at both the permissive and the nonpermissive temperatures. We conclude that the five VV mutants affect the 132-kDa subunit of the VV RNA polymerase. Additional genetic experiments demonstrate intragenic complementation between ts 62 and three other members of this complementation group, ts 27, ts 29, and ts 32.

Authors
Hooda-Dhingra, U; Patel, DD; Pickup, DJ; Condit, RC
MLA Citation
Hooda-Dhingra, U, Patel, DD, Pickup, DJ, and Condit, RC. "Fine structure mapping and phenotypic analysis of five temperature-sensitive mutations in the second largest subunit of vaccinia virus DNA-dependent RNA polymerase." Virology 174.1 (January 1990): 60-69.
PMID
2294648
Source
pubmed
Published In
Virology
Volume
174
Issue
1
Publish Date
1990
Start Page
60
End Page
69

Transcription of a poxvirus early gene is regulated both by a short promoter element and by a transcriptional termination signal controlling transcriptional interference.

The promoter region of an early gene (38K gene) of cowpox virus has been characterized by deletion and linker scanning mutational analyses. Modified versions of this promoter region were placed into the genome of vaccinia virus, and their transcriptional efficiencies were assessed by quantifying RNAs transcribed from these sequences. These analyses showed that the sequences in the region between 33 and 4 base pairs upstream of the transcriptional start site affect the efficiency of transcription from this promoter. Linker scanning mutations in the -27 to -10 region inhibited transcription. This region contains the sequence 5'-GAAAATATATT-3', which is present in at least two other early genes in the same positions (-21 to -11) relative to the transcriptional start sites of these genes. Elements of this sequence are similarly positioned in the promoter regions of several other poxvirus genes, suggesting that this sequence represents a transcriptional control element of at least a subset of poxvirus genes. The -8 to -2 sequence (5'-TTTTTAT-3') contains a transcriptional termination signal. Mutation of this sequence had two separate effects: (i) it reduced the efficiency of transcription from the promoter by approximately 30%, and (ii) it prevented this sequence from terminating the transcription from upstream genes. When overlapping transcription from upstream genes was not prevented by a termination signal present either within the 38K promoter or upstream of the promoter, transcription from this promoter was reduced by about 30%. This indicates that transcriptional termination has a role in the regulation of viral gene expression by controlling transcriptional interference.

Authors
Ink, BS; Pickup, DJ
MLA Citation
Ink, BS, and Pickup, DJ. "Transcription of a poxvirus early gene is regulated both by a short promoter element and by a transcriptional termination signal controlling transcriptional interference." J Virol 63.11 (November 1989): 4632-4644.
PMID
2795715
Source
pubmed
Published In
Journal of virology
Volume
63
Issue
11
Publish Date
1989
Start Page
4632
End Page
4644

Inhibition of an inflammatory response is mediated by a 38-kDa protein of cowpox virus.

The Brighton Red (BR) strain of cowpox virus induces a flat, bright red pock on the chorioallantoic membrane (CAM) of the 12-day-old chick embryo. In contrast, mutants with a deleted 38K gene (which is located 31 to 32 kb from the right-hand end of the virus genome) induced a raised, white, and opaque pock. During the first 24-hr p.i., both CPV-BR and the 38K deletion mutants replicated similarly in the CAM of the chick embryo, as indicated by immunocytochemical detection of similar amounts of virus antigen. By 48 hr p.i., the pocks induced by the mutant and CPV-BR are strikingly different. The pocks induced by the 38K deletion mutants were infiltrated by large numbers of heterophils and macrophages, which correlated with a reduction in the levels of virus antigen and virus infectivity. The CPV-BR pock had an absence of inflammatory cells and increased levels of virus antigen and infectivity. By 72 hr p.i., many of the pocks induced by the mutant were undergoing resolution of the virus infection, as indicated by further decrease of virus antigen and visible signs of healing, whereas CPV-BR pocks continued to be a site of active viral replication. These data are consistent with a model where this 38-kDa protein directly or indirectly inhibits the generation of chemotactic molecules which are elicited during virus replication in the CAM or, alternatively, blocks the interaction of these molecules with cells of the host inflammatory response.

Authors
Palumbo, GJ; Pickup, DJ; Fredrickson, TN; McIntyre, LJ; Buller, RM
MLA Citation
Palumbo, GJ, Pickup, DJ, Fredrickson, TN, McIntyre, LJ, and Buller, RM. "Inhibition of an inflammatory response is mediated by a 38-kDa protein of cowpox virus." Virology 172.1 (September 1989): 262-273.
PMID
2773318
Source
pubmed
Published In
Virology
Volume
172
Issue
1
Publish Date
1989
Start Page
262
End Page
273

The second-largest subunit of the poxvirus RNA polymerase is similar to the corresponding subunits of procaryotic and eucaryotic RNA polymerases.

We have characterized the poxvirus gene encoding the second-largest subunit of the viral DNA-dependent RNA polymerase. This gene, designated rpo132, is located in the HindIII A fragment of the DNA of the Brighton Red strain of cowpox virus. A similar gene is located in the corresponding position in the HindIII A fragment of the DNA of the Western Reserve strain of vaccinia virus. The rpo132 gene is transcribed throughout the viral multiplication cycle. It has two transcriptional start sites; one is operative at late times only, and the other (80 base pairs downstream) is operative both at early times and at late times. Neither early nor late transcripts originating from the latter RNA start site contain long 5'-terminal poly(A) sequences. The rpo132 gene has the capacity to encode primary gene products of two types. The RNA transcripts whose 5' ends correspond to the early RNA start site can encode a 133-kilodalton (kDa) protein. The RNA transcripts whose 5' ends correspond to the early RNA start site can encode a 132-kDa protein. Transcripts of the latter type are more abundant, suggesting that the 132-kDa protein is the major primary product of this gene. The predicted amino acid sequences of both gene products share extensive similarities with the amino acid sequences of the second-largest subunits of the following enzymes: the RNA polymerase of Escherichia coli, the RNA polymerase II of Saccharomyces cerevisiae, and the RNA polymerase II of Drosophila melanogaster. This result provides further evidence of relatedness between multisubunit DNA-dependent RNA polymerases.

Authors
Patel, DD; Pickup, DJ
MLA Citation
Patel, DD, and Pickup, DJ. "The second-largest subunit of the poxvirus RNA polymerase is similar to the corresponding subunits of procaryotic and eucaryotic RNA polymerases." J Virol 63.3 (March 1989): 1076-1086.
PMID
2915377
Source
pubmed
Published In
Journal of virology
Volume
63
Issue
3
Publish Date
1989
Start Page
1076
End Page
1086

THE 2ND-LARGEST SUBUNIT OF THE POXVIRUS RNA-POLYMERASE IS SIMILAR TO THE CORRESPONDING SUBUNITS OF PROCARYOTIC AND EUKARYOTIC RNA-POLYMERASES

Authors
PATEL, DD; PICKUP, DJ
MLA Citation
PATEL, DD, and PICKUP, DJ. "THE 2ND-LARGEST SUBUNIT OF THE POXVIRUS RNA-POLYMERASE IS SIMILAR TO THE CORRESPONDING SUBUNITS OF PROCARYOTIC AND EUKARYOTIC RNA-POLYMERASES." JOURNAL OF VIROLOGY 63.3 (March 1989): 1076-1086.
Source
wos-lite
Published In
Journal of virology
Volume
63
Issue
3
Publish Date
1989
Start Page
1076
End Page
1086

Book review: 'The orthopoxviruses' (F. Fenner, R. Wittek, and K.R. Dumbell)

Authors
Pickup, DJ
MLA Citation
Pickup, DJ. "Book review: 'The orthopoxviruses' (F. Fenner, R. Wittek, and K.R. Dumbell)." Cell 59.4 (January 1, 1989): 589-590.
Source
manual
Published In
Cell
Volume
59
Issue
4
Publish Date
1989
Start Page
589
End Page
590

High-level synthesis of biologically active reovirus protein sigma 1 in a mammalian expression vector system.

The reovirus cell-attachment protein, which modulates tissue tropism and the nature of the antiviral immune response, is protein sigma 1. This protein is present in reovirus particles in the form of 12 tetramers anchored in the projections or spikes. It is formed in infected cells in very small amounts only. In order to produce larger amounts of it that are necessary for studying its properties in detail, we have cloned it into a mammalian expression vector system that consists of the very strong cowpox virus A-type inclusion body protein gene promoter inserted into the vaccinia virus genome. In cells infected with the resultant recombinant vaccina virus, protein sigma 1 is formed in large amounts (2-3 mg/10(9) cells). We have isolated the native protein and shown that it exists as tetramers that possess cell-binding activity, hemagglutinating activity, and functional epitopes recognized by both polyvalent antisera and monoclonal antibodies.

Authors
Banerjea, AC; Brechling, KA; Ray, CA; Erikson, H; Pickup, DJ; Joklik, WK
MLA Citation
Banerjea, AC, Brechling, KA, Ray, CA, Erikson, H, Pickup, DJ, and Joklik, WK. "High-level synthesis of biologically active reovirus protein sigma 1 in a mammalian expression vector system." Virology 167.2 (December 1988): 601-612.
PMID
3201754
Source
pubmed
Published In
Virology
Volume
167
Issue
2
Publish Date
1988
Start Page
601
End Page
612

A poxvirus-derived vector that directs high levels of expression of cloned genes in mammalian cells.

High levels of expression of cloned genes have been obtained in mammalian cells by using poxvirus-derived insertion/expression vectors. These vectors employ the cis-acting element (CAE I) that directs the transcription of one of the most strongly expressed genes of cowpox virus. This gene (the 160K gene) encodes the 160-kDa protein that is the major component of the A-type cytoplasmic inclusions. Its counterpart in vaccinia virus (VV) is the 94K gene contained in the HindIII A fragment of the viral DNA. Two insertion vectors have been constructed; each is designed to allow cloned genes to be placed immediately downstream of a modified version of CAE I within a poxvirus genome. One vector, p1200, enables the CAE I-cloned-gene constructs to be inserted into the thymidine kinase gene of VV. This vector was used to create a VV recombinant that directed expression of the chloramphenicol acetyltransferase (CAT) gene. The other vector, p2101, enables the CAE I-cloned-gene constructs to be inserted into the VV 94K gene. The prototype of this vector was used to create a VV recombinant that directed expression of a hybrid CAT-lacZ gene. Infection of cultured human cells with these recombinants led to high levels of synthesis of either the CAT gene product or the CAT-lacZ gene product. Each of these proteins was produced in quantities that were easily detected by Coomassie blue staining of total cell proteins resolved by polyacrylamide gel electrophoresis. We estimate that these vectors are capable of directing the synthesis of milligram amounts of gene product per 10(9) mammalian cells.

Authors
Patel, DD; Ray, CA; Drucker, RP; Pickup, DJ
MLA Citation
Patel, DD, Ray, CA, Drucker, RP, and Pickup, DJ. "A poxvirus-derived vector that directs high levels of expression of cloned genes in mammalian cells." Proc Natl Acad Sci U S A 85.24 (December 1988): 9431-9435.
PMID
2849105
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
85
Issue
24
Publish Date
1988
Start Page
9431
End Page
9435

Virulence genes of poxviruses and reoviruses.

Identification of viral genes that specify virulence, however defined, is of critical importance for the design of viral vaccines. In particular, the targeted development not only of avirulent vaccine strains but also of viruses to be used as carriers for foreign genes depends on the inactivation or deletion of genes that harm the host. This paper illustrates approaches to identifying viral pathogenesis genes in two model systems involving cowpox virus and reovirus, respectively.

Authors
Joklik, WK; Pickup, DJ; Patel, DD; Moody, MD
MLA Citation
Joklik, WK, Pickup, DJ, Patel, DD, and Moody, MD. "Virulence genes of poxviruses and reoviruses." Vaccine 6.2 (April 1988): 123-128. (Review)
PMID
2838983
Source
pubmed
Published In
Vaccine
Volume
6
Issue
2
Publish Date
1988
Start Page
123
End Page
128

Messenger RNAs of a strongly-expressed late gene of cowpox virus contain 5'-terminal poly(A) sequences.

We have identified and characterized one of the most strongly-expressed genes of cowpox virus (CPV). This is the gene encoding the major protein component of the A-type inclusion bodies produced by this virus. This gene (designated the 160K gene) is transcribed late during the infection. Analyses of its mRNAs showed that these late RNAs, unlike all other characterized late mRNAs of poxviruses, are uniform in length. However, the most remarkable feature of the mRNAs of the 160K gene is the structure of their 5'-termini. Most of these mRNAs have 5'-terminal poly(A) sequences containing 5-21 residues. Furthermore, these 5'-terminal poly(A) sequences are not complementary to the corresponding region of the template strand of the viral DNA. Instead, the nucleotide sequences of the mRNA and the viral DNA diverge at the site of the three As in the sequence 5'-TAAATG-3' containing the gene's initiation codon. Consequently, the poly(A) provides the leader sequences of these mRNAs. These unusual 5'-terminal structures suggest that the late mRNAs of pox-virus genes are generated by a novel process.

Authors
Patel, DD; Pickup, DJ
MLA Citation
Patel, DD, and Pickup, DJ. "Messenger RNAs of a strongly-expressed late gene of cowpox virus contain 5'-terminal poly(A) sequences." EMBO J 6.12 (December 1, 1987): 3787-3794.
PMID
2828037
Source
pubmed
Published In
EMBO Journal
Volume
6
Issue
12
Publish Date
1987
Start Page
3787
End Page
3794

Tandemly repeated sequences are present at the ends of the DNA of raccoonpox virus.

The DNA of raccoonpox virus (RCN) has been characterized by restriction enzyme analysis. DNA hybridization studies showed that all HindIII fragments of the 215-kbp RCN DNA share some nucleotide sequence similarity with fragments of the DNA of cowpox virus (CPV). This information was used to construct a HindIII restriction map of the RCN DNA. The nucleotide sequence of the 2.2-kbp Sal 1 end fragment of the RCN DNA has been determined from a cloned copy of the HindIII O fragment. Of this 2.2-kb region 75% consists of short, tandemly repeated sequences. It does not contain any open reading frames capable of encoding polypeptide chains of more than 62 amino acids. There are six related types of repeated sequence, and these are arranged into two separate sets, each flanked by nonrepeated sequences. The nucleotide sequences of both repeated and nonrepeated sequences within this Sal 1 fragment are extremely similar to those of the Sal 1-generated end fragments of the DNas of CPV and vaccinia virus. The arrangements of the repeated and nonrepeated sequences are also similar in the DNAs of these three viruses. In contrast, the remainder of the RCN DNA is markedly different from the DNAs of other orthopoxviruses. The high degree of similarity between the ends of the RCN DNA and the ends of the other orthopoxvirus DNAs suggest that the complex arrays of repeated and nonrepeated sequences have been conserved because they have a role in virus multiplication.

Authors
Parsons, BL; Pickup, DJ
MLA Citation
Parsons, BL, and Pickup, DJ. "Tandemly repeated sequences are present at the ends of the DNA of raccoonpox virus." Virology 161.1 (November 1987): 45-53.
PMID
2823468
Source
pubmed
Published In
Virology
Volume
161
Issue
1
Publish Date
1987
Start Page
45
End Page
53

Hemorrhage in lesions caused by cowpox virus is induced by a viral protein that is related to plasma protein inhibitors of serine proteases.

Several recombinant cowpox viruses were constructed and used to identify a viral gene that controls the production of hemorrhage in lesions caused by the Brighton Red strain of cowpox virus (CPV-BR). This gene is located in the KpnD fragment of CPV-BR DNA, between 31 and 32 kilobases from the end of the genome. This position corresponds well with that predicted from analyses of the DNA structures of spontaneously generated deletion mutants. The gene responsible for hemorrhage encodes a 38-kDa protein that is one of the most abundant early gene products. The 11-base-pair sequence GAAAATATATT present 84 base pairs upstream of its coding region is also present upstream of three other early genes of vaccinia virus; therefore, this sequence may be involved in the regulation of transcription. There is extensive similarity between the predicted amino acid sequence of the 38-kDa protein and the amino acid sequences of several plasma proteins that are inhibitors of various serine proteases involved in blood coagulation pathways. This suggests that the viral protein may possess a similar biological activity, which may enable it to effect hemorrhage by inhibiting one or more of the serine proteases involved in the host's normal processes of blood coagulation and wound containment.

Authors
Pickup, DJ; Ink, BS; Hu, W; Ray, CA; Joklik, WK
MLA Citation
Pickup, DJ, Ink, BS, Hu, W, Ray, CA, and Joklik, WK. "Hemorrhage in lesions caused by cowpox virus is induced by a viral protein that is related to plasma protein inhibitors of serine proteases." Proc Natl Acad Sci U S A 83.20 (October 1986): 7698-7702.
PMID
3532120
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
83
Issue
20
Publish Date
1986
Start Page
7698
End Page
7702

Isolation of cowpox virus A-type inclusions and characterization of their major protein component.

A-type inclusions (ATI)2 are large well-defined structures that appear in the cytoplasm during the late stages of the multiplication cycles of many poxviruses. The ATIs produced by the CPRC1 strain of cowpox virus in strain 143 human osteosarcoma cells have been isolated and characterized. These inclusions which can be recovered in large amounts (about 20 mg per 10(9) cells), appear to consist entirely of a single protein species that has a molecular weight of 160,000. It is a late protein, stable in infected cells, and during the late stages of the multiplication cycle it is one of the most abundantly synthesized proteins. Very similar ATIs composed of a serologically closely related protein of the same size are formed in cells infected with raccoonpox virus. In cells infected with vaccinia virus strain WR much smaller inclusions are formed; further, in such cells large amounts of a late protein with a molecular weight of 94,000 are produced that is also closely related to the cowpox virus ATI protein.

Authors
Patel, DD; Pickup, DJ; Joklik, WK
MLA Citation
Patel, DD, Pickup, DJ, and Joklik, WK. "Isolation of cowpox virus A-type inclusions and characterization of their major protein component." Virology 149.2 (March 1986): 174-189.
PMID
3456179
Source
pubmed
Published In
Virology
Volume
149
Issue
2
Publish Date
1986
Start Page
174
End Page
189

Spontaneous deletions and duplications of sequences in the genome of cowpox virus.

Examination of the genomes of 10 white-pock variants of cowpox virus strain Brighton red (CPV-BR) revealed that 9 of them had lost 32 to 38 kilobase pairs (kbp) from their right-hand ends and that the deleted sequences had been replaced by inverted copies of regions from 21 to 50 kbp long from the left-hand end of the genome. These variants thus possess inverted terminal repeats (ITRs) from 21 to 50 kbp long; all are longer than the ITRs of CPV-BR (10 kbp). The 10th variant is a simple deletion mutant that has lost the sequences between 32 and 12 kbp from the right-hand end of the genome. The limits of the inner ends of the observed deletions (between 32 and 38 kbp from the right-hand end of the CPV-BR genome) appear to be defined by the location of the nearest essential gene on the one hand and the location of the gene that encodes "pock redness" on the other. The genomes of the deletion/duplication white-pock variants appear to have been generated either by single crossover recombinational events between two CPV-BR genomes aligned in opposite directions or by the nonreciprocal transfer of genetic information. The sites where such recombination/transfer occurred were sequenced in four variants. In all of them, the sequences adjacent to such sites show no sequence homology or any other unusual structural feature. The analogous sites at the internal ends of the two ITRs of CPV-BR also were sequenced and also show no unusual features. It is likely that the ITRs of CPV-BR and of its white-pock variants, and probably those of other orthopox-virus genomes, arise as a result of nonhomologous recombination or by random nonreciprocal transfer of genetic information.

Authors
Pickup, DJ; Ink, BS; Parsons, BL; Hu, W; Joklik, WK
MLA Citation
Pickup, DJ, Ink, BS, Parsons, BL, Hu, W, and Joklik, WK. "Spontaneous deletions and duplications of sequences in the genome of cowpox virus." Proc Natl Acad Sci U S A 81.21 (November 1984): 6817-6821.
PMID
6093123
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
81
Issue
21
Publish Date
1984
Start Page
6817
End Page
6821

Spontaneous deletions and duplications of sequences in the genome of cowpox virus

Examination of the genomes of 10 white-pock variants of cowpox virus strain Brighton red (CPV-BR) revealed that 9 of them had lost 32 to 38 kilobase pairs (kbp) from their right-hand ends and that the deleted sequences had been placed by inverted copies of regions from 21 to 50 kbp long from the left-hand end of the genome. These variants thus possess inverted terminal repeats (ITRs) from 21 to 50 kbp long; all are longer than the ITRs of CPV-BR (10 kbp). The 10th variant is a simple deletion mutant that has lost the sequences between 32 and 12 kbp from the right-hand end of the genome. The limits of the inner ends of the observed deletions (between 32 and 38 kbp from the right-hand end of the CPV-BR genome) appear to be defined by the location of the nearest essential gene on the one hand and the location of the gene that encodes 'pock redness' on the other. The genomes of the deletion/duplication white-pock variants appear to have been generated either by single crossover recombinational events between two CPV-BR genomes aligned in opposite directions or by the nonreciprocal transfer of genetic information. The sites where such recombination/transfer occurred were sequenced in four variants. In all of them, the sequences adjacent to such sites show no sequences homology or any other unusual structure feature. The analogous sites at the internal ends of the two ITRs of CPV-BR also were sequenced and also show no unusual features. It is likely that the ITRs of CPV-BR and of its white-pock variants, and probably those of other orthopox-virus genomes, arise as a result of nonhomologous recombination or by random nonreciprocal transfer of genetic information.

Authors
Pickup, DJ; Ink, BS; Parsons, BL; Hu, W; Joklik, WK
MLA Citation
Pickup, DJ, Ink, BS, Parsons, BL, Hu, W, and Joklik, WK. "Spontaneous deletions and duplications of sequences in the genome of cowpox virus." Proceedings of the National Academy of Sciences of the United States of America 81.21 I (1984): 6817-6821.
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
81
Issue
21 I
Publish Date
1984
Start Page
6817
End Page
6821
DOI
10.1073/pnas.81.21.6817

Cloning of the terminal loop of vaccinia virus DNA.

Authors
Pickup, DJ; Bastia, D; Joklik, WK
MLA Citation
Pickup, DJ, Bastia, D, and Joklik, WK. "Cloning of the terminal loop of vaccinia virus DNA." Virology 124.1 (January 15, 1983): 215-217.
PMID
6297156
Source
pubmed
Published In
Virology
Volume
124
Issue
1
Publish Date
1983
Start Page
215
End Page
217

Sequence of terminal regions of cowpox virus DNA: arrangement of repeated and unique sequence elements.

One terminal EcoRI fragment of the genome of cowpox virus (CPV) strain Brighton red has been cloned in plasmid pBR325, and the nucleotide sequence of the 2,725-base-pair Sal I fragment corresponding to that at the end of the viral genome has been determined. The fragment consists of three unique sequence regions flanking two sets of repeated sequence. The repeated sequence sets are composed of four types of subunits, the majority of which are arranged in higher-order repeat units. The subunits are themselves closely related; two are subsets of a third, whereas the fourth is a recombinant of the first two. The fragment possesses no long open reading frames (maximal coding potential, 65 amino acids). The sequence of this CPV DNA Sal I fragment is compared with that of the corresponding fragment of vaccinia virus WR DNA [Baroudy, B. M., Venkatesan, S. & Moss, B. (1982) Cell 28, 315-324; Venkatesan, S., Baroudy, B. M. & Moss, B. (1981) Cell 25, 805-813]. Two of the unique sequence regions of the two viruses are related to the extent of 96%, and the third contains at least one sequence of 112 residues that is 98% homologous. As for the repeated sequence sets, those of vaccinia virus are composed of only two, rather than four, types of subunit, one of which is identical to one of the CPV subunits, whereas the other differs from another CPV subunit by only three mismatches and one deletion. However, the arrangement of subunits in the two viruses is different, that in vaccinia virus DNA being simpler. Both subunits as well as repeat units probably arose as a result of unequal crossover.

Authors
Pickup, DJ; Bastia, D; Stone, HO; Joklik, WK
MLA Citation
Pickup, DJ, Bastia, D, Stone, HO, and Joklik, WK. "Sequence of terminal regions of cowpox virus DNA: arrangement of repeated and unique sequence elements." Proc Natl Acad Sci U S A 79.23 (December 1982): 7112-7116.
PMID
6961398
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
79
Issue
23
Publish Date
1982
Start Page
7112
End Page
7116

Sequence at both termini of the 10 genes of reovirus serotype 3 (strain Dearing).

Authors
Antczak, JB; Chmelo, R; Pickup, DJ; Joklik, WK
MLA Citation
Antczak, JB, Chmelo, R, Pickup, DJ, and Joklik, WK. "Sequence at both termini of the 10 genes of reovirus serotype 3 (strain Dearing)." Virology 121.2 (September 1982): 307-319.
PMID
6927854
Source
pubmed
Published In
Virology
Volume
121
Issue
2
Publish Date
1982
Start Page
307
End Page
319

Sequence of terminal regions of cowpox virus DNA: Arrangement of repeated and unique sequence elements

One terminal EcoRI fragment of the genome of cowpox virus (CPV) strain Brighton red has been cloned in plasmid pBR325, and the nucleotide sequence of the 2,725-base-pair Sal I fragment corresponding to that at the end of the viral genome has been determined. The fragment consists of three unique sequence regions flanking two sets of repeated sequence. The repeated sequence sets are composed of four types of subunits, the majority of which are arranged in higher-order repeat units. The subunits are themselves closely related; two are subsets of a third, whereas the fourth is a recombinant of the first two. The fragment possesses no long open reading frames (maximal coding potential, 65 amino acids). The sequence of this CP DNA SalI fragment is compared with that of the corresponding fragment of vaccinia virus WR DNA. Two of the unique sequence regions of the two viruses are related to the extent of 96%, and the third contains at least one sequence of 112 residues that is 98% homologous. As for the repeated sequence sets, those of vaccinia virus are composed of only two, rather than four, types of subunit, one of which is identical to one of the CPV subunits, whereas the other differs from another CPV subunit by only three mismatches and one deletion. However, the arrangement of subunits in the two viruses is different, that in vaccinia virus DNA being simpler. Both subunits as well as repeat units probably arose as a result of unequal cross-over.

Authors
Pickup, DJ; Bastia, D; Stone, HO; Joklik, WK
MLA Citation
Pickup, DJ, Bastia, D, Stone, HO, and Joklik, WK. "Sequence of terminal regions of cowpox virus DNA: Arrangement of repeated and unique sequence elements." Proceedings of the National Academy of Sciences of the United States of America 79.23 I (1982): 7112-7116.
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
79
Issue
23 I
Publish Date
1982
Start Page
7112
End Page
7116
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